I gratefully acknowledge the support of the Slovenian Research Agency, grant no. P4-133.

1. Basic histology for honeybee research
<ol>
	<li>Dissection of honeybee tissue 
	NOTE: For the dissection of worker bees, use a dissecting microscope with an LED light source. The most useful magnification is ~20x.
	<ol>
		<li>Manipulation and dissection
		<ol>
			<li>Carefully take a worker bee with forceps and put it on ice (or into the freezer at -20 &#176;C) for 2 min to immobilize it22. Pin the bee on the Petri dish diagonally through the uppermost back portion of the thorax twice, from left to right and from right to left.</li>
			<li>Pour insect saline to cover the body. Place the Petri dish under the microscope, focus, and adjust.</li>
			<li>Prepare the instruments (see the Table of Materials).</li>
		</ol>
		</li>
		<li>Dissection of midgut
		<ol>
			<li>Start with the abdomen by inserting one point of the scissors under the tergite A5 (Figure 1) in the center of the right side of the bee body. Cut to the tergite A2.</li>
			<li>Keep the inner blade of the scissors parallel with the side of the body to avoid damaging the internal organs. Turn the scissors left and make one cut; turn right and make another cut. Gently open the left part of the abdomen and pin it. Repeat on the other side.</li>
			<li>Using forceps with one hand, gently pull the honeybee stomach upward, and with scissors in the other hand, cut at the very end of the esophagus. Pull the stomach and midgut away from the abdomen and cut at the rectum. Use a pipette with insect saline solution and remove any feces or parts of the tissue.</li>
		</ol>
		</li>
		<li>Dissection of HPGs
		<ol>
			<li>Immobilize a worker bee on ice as described in step 1.1.1. Cut the head off and place it on the smaller plate with the antennae facing up. Secure the head with two pins: one through the left compound eye and the second through the right compound eye.</li>
			<li>Make a cut across the first compound eye on the inner side of the pins, continue to the labrum, and then make another cut on the other side across the second compound eye (Figure 2).</li>
			<li>Cut off the antennae. Lift off the mask and cut where still attached. Take the forceps and carefully remove the glands together with the brain and part of the compound eyes.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Fixation, dehydration, and paraffin embedding 
	NOTE: Wear protective gloves.
	<ol>
		<li>Place the tissue in penicillin bottles, filled 3/4 with 10% formalin. Keep in a refrigerator at 4 &#176;C.</li>
		<li>After 24 h, dehydrate the tissue in a series of alcohols: 70%, 80%, 90%, 100%, for 1 h each, 100% 2-propanol for 1 h, 100% 2-propanol for 12 h, and finally 100% 2-propanol for 1 h.</li>
		<li>Place the tissue in histocassettes; mark and place them in the glass chambers with 2-propanol and paraffin (1:1) in an incubator at 60 &#176;C for 24 h.</li>
		<li>Move the histocassettes to another chamber with paraffin (I.) for another 24 h. Repeat the procedure with fresh paraffin twice more (II. and III.), both for 24 h.</li>
		<li>Finally, prepare the mounting station and start embedding the tissue into wax.
		<ol>
			<li>Open each histocassette and remove the cover. Fill the mold with wax and carefully put the tissue with warm forceps in the middle of the mold.</li>
			<li>Place the histocassette on the mold and slightly cover it with wax. Immediately place the mold on the cold surface of the mounting station for a few seconds, then place it on the cold plate for a few minutes until the wax hardens and separates from the mold together and the histocassette.</li>
		</ol>
		</li>
		<li>Store the finished samples in a box, away from dust and heat.</li>
		<li>Cut 4 &#181;m thin sections on a microtome: first, two sections attached to each other and then one separately. Transfer the sections with forceps and let them float on distilled water (42 &#176;C), then collect them on clean slides by placing two sections together on the left side of the objective glass and the third one on the right side, remaining distinctly separate. Leave the marked slides overnight on the heating device and finally store them in a box dedicated for histology samples.</li>
	</ol>
	</li>
	<li>Dewaxing and rehydration 
	NOTE: Wear protective gloves.
	<ol>
		<li>Prepare nine Coplin jars and put the sections into a series of clearing agents (I., II., III.) for 5 min each.</li>
		<li>Put into 2-propanol, ethanol 96% (I., II.), alcohol 90% and 80%, and distilled water for 3 min each.</li>
	</ol>
	</li>
	<li>Dyeing with hematoxylin and eosin 
	NOTE: Wear protective gloves.
	<ol>
		<li>Prepare six Coplin jars.</li>
		<li>For hematoxylin and eosin (H&#38;E) staining, put the dewaxed, rehydrated sections in hematoxylin for 5 min, then carefully place them under the running tap water for 2 min. Then put them into distilled water for 1 min and eosin for 4 min (for eosin, the Coplin jar is not necessary).</li>
		<li>Place the slides in ethanol 96% for 1 min, then 2-propanol for 2 min, and finally into the clearing agent for 2 min.</li>
		<li>Add mounting medium and a cover glass and let them dry. Observe under a light microscope.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64141/64141fig01.jpg" /> 
Figure 1: Dorsal view of honeybee body. A1-A7 tergites. The detailed instructions on honeybee dissection can be found in Carreck et al.24. <a href="https://www.jove.com/files/ftp_upload/64141/64141fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64141/64141fig02v2.jpg" /> 
Figure 2: Dorsal view of HPGs, parts of compound eyes attached to the brain (not visible). A young worker bee aged 5 to 6 days has plump and creamy white HPGs. The acini are located on the brain and fill the head area with branches reaching the back of the brain. In foraging bees, these glands are greatly shrunken and leave only thin thread-like remains. For this reason, it is better to remove glands together with the brain to make it easier in further procedures to avoid losing the tissue. Scale bar = 500 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64141/64141fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Cell death detection in tissue sections
<ol>
	<li>Apoptosis detection kit (Assay A) 
	NOTE: Follow the manufacturer&#39;s protocol (see the Table of Materials).
	<ol>
		<li>Prepare the Coplin jars.</li>
		<li>After dewaxing and rehydration (see step 1.3), immerse the slides in 0.85% NaCl solution, and then in phosphate-buffered saline (PBS) (5 min).</li>
		<li>Put the slides in 4% paraformaldehyde 2 x 15 min.</li>
		<li>Place the slides flat in the container and add 100 &#181;L of a Proteinase K (20 &#181;g/mL) solution, then leave them for 10-30 min.</li>
		<li>Place the slides in PBS (5 min).</li>
		<li>Place the slides in 4% paraformaldehyde in PBS (5 min).</li>
		<li>Immerse the slides in PBS (2 x 5 min).</li>
		<li>Place the slides flat in the container, add 100 &#181;L of equilibration buffer, and leave them for 5-10 min.</li>
		<li>Add 100 &#181;L of TdT reaction mix. Put paper towels inside the container, around the slides, moisten the towels with water, and then cover with plastic wrap. Incubate slides for 60 min at 37 &#176;C.</li>
		<li>Place the slides back in the staining rack and immerse in 2x saline-sodium citrate (SSC) for 15 min.</li>
		<li>Immerse the slides 3 x 5 min in PBS, then in 0.3% hydrogen peroxide for 3-5 min, and then in PBS again, 3 x 5 min.</li>
		<li>Again, place the slides flat in the container, add 100 &#181;L of Streptavidin HRP (horseradish peroxidase), and leave for 30 min (cover with plastic wrap).</li>
		<li>Immerse the slides 3 x 5 min in PBS.</li>
		<li>Place the slides flat in the container and add 100 &#181;L of 3,3&#39;-diaminobenzidine (DAB) solution. Look for a light brown background.</li>
		<li>Return the slides to the rack and wash them several times in water (double-distilled).</li>
		<li>Mount the slides under glass coverslips in mounting medium and leave flat to dry.</li>
		<li>Observe under a light microscope.</li>
	</ol>
	</li>
	<li>Apoptosis detection kit (Assay B) 
	NOTE: Follow the manufacturer&#39;s protocol (see the Table of Materials).
	<ol>
		<li>Prepare Coplin jars.</li>
		<li>Prepare Proteinase K (20 &#181;g/mL diluted in PBS).</li>
		<li>After dewaxing and rehydrating the sections (step 1.3), place the slides in PBS for 5 min.</li>
		<li>Place the slides flat in the container and add Proteinase K (20 &#181;g/mL, 60 &#181;L per 5 cm&#178; specimen).</li>
		<li>Wash the slides 2 x 2 min in distilled water.</li>
		<li>Quench in endogenous peroxidase (in 3% hydrogen peroxidase) at room temperature.</li>
		<li>Rinse the slides 2 x 5 min with PBS or water.</li>
		<li>Place the slides flat in the container and apply equilibration buffer (75 &#181;L/5 cm2) for 10 s at room temperature.</li>
		<li>Carefully wipe around the tissue.</li>
		<li>Add the TdT enzyme (terminal deoxynucleotidyl transferase) to each section and incubate in a humidified chamber for 1 h at 37 &#176;C. Put paper towels inside the tray, around the slides, moisten the towels with water, and cover them with plastic wrap.</li>
		<li>After incubation, put the specimens in the rack and leave them in stop/wash buffer (10 min).</li>
		<li>Warm the anti-digoxigenin conjugate to room temperature.</li>
		<li>Wash the slides in PBS (3 x 1 min).</li>
		<li>Carefully wipe off around the tissue.</li>
		<li>Add two drops of Anti-Digoxigenin-Peroxidase conjugate (65 &#181;L/5 cm&#178;) to the sections and incubate for 30 min in a humidified container.</li>
		<li>After washing in PBS 4 x 2 min, prepare working-strength peroxidase substrate, and gently tap off excess liquid and aspirate around the section.</li>
		<li>Cover the sections with peroxidase substrate (75 &#181;L/5 cm&#178;) and stain for 5 min. Place a slide under the microscope and determine the optimal staining time.</li>
		<li>Wash the slides in a staining rack in distilled water (3 x 1 min).</li>
		<li>Incubate the slides in distilled water for 5 min.</li>
		<li>Counterstain using hematoxylin for 2 min.</li>
		<li>Place the slide under running tap water for 3 min.</li>
		<li>Wash the slide in distilled water.</li>
		<li>Mount the slides under glass coverslips in mounting medium and leave flat to dry.</li>
		<li>Observe under a light microscope.</li>
	</ol>
	</li>
	<li>Apoptosis detection kit (Assay C) 
	NOTE: Follow the manufacturer&#39;s protocol (see the Table of Materials).
	<ol>
		<li>Prepare the Coplin jars.</li>
		<li>Dewax and rehydrate the tissue sections (see step 1.3).</li>
		<li>Incubate the tissue with Proteinase K (15-30 min at 37 &#176;C).</li>
		<li>Place the slides back on the rack and rinse 2x in PBS.</li>
		<li>Cover with 50 &#181;L of &#39;TUNEL reaction mixture&#39;. Place wet paper towels inside the container, cover them with plastic wrap, and leave them for 60 min at 37 &#176;C.</li>
		<li>Rinse 3x with PBS.</li>
		<li>Place the slides in the container and dry the area around the tissue sample.</li>
		<li>Add 50 &#181;L of Converter-AP to the sample and incubate in a humidified container for 30 min at 37 &#176;C.</li>
		<li>Rinse 3x in PBS.</li>
		<li>Add 50-100 &#181;L of substrate solution and leave for 10 min in the dark. 
		NOTE: Observe the staining under a light microscope.</li>
		<li>Rinse the slides 3x with PBS.</li>
		<li>Counterstain by transferring sections into hematoxylin for 2 min and then carefully rinse in running tap water for 5 min.</li>
		<li>Mount the slides under glass coverslips in an aqueous mounting medium and leave them flat to dry.</li>
		<li>Observe under a light microscope. Assess the affected (positive) cells by counting 70 to 100 cells in each sample of the midgut or HPGs under a light microscope.</li>
	</ol>
	</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmed+cell+death+in+the+salivary+gland+of+the+blow+fly+Calliphora+vomitoria.">Programmed cell death in the salivary gland of the blow fly Calliphora vomitoria.</a> Microscopy Research Techniques. 34, 202-207 (1996).</li><li>D'Arcy, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death:+a+review+of+the+major+forms+of+apoptosis,+necrosis+and+autophagy.">Cell death: a review of the major forms of apoptosis, necrosis and autophagy.</a> Cell Biology International. 43 (6), 582-592 (2019).</li><li>Matylevitch, N. 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D., Ciocca, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+detection+of+apoptotic+cells+using+a+modified+in+situ+TUNEL+technique.">Improved detection of apoptotic cells using a modified in situ TUNEL technique.</a> Journal of Histochemistry and Cytochemistry. 47 (6), 837-839 (1999).</li><li>Gregorc, A., Bowen, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmed+cell+death+in+the+honeybee+(Apis+mellifera+L.)+larvae+midgut.">Programmed cell death in the honeybee (Apis mellifera L.) larvae midgut.</a> Cell Biology International. 21 (3), 151-158 (1997).</li><li>Gregorc, A., Poga&#269;nik, A., Bowen, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+in+honey+bee+(Apis+mellifera)+larvae+treated+with+oxalic+or+formic+acid.">Cell death in honey bee (Apis mellifera) larvae treated with oxalic or formic acid.</a> Apidologie. 35 (5), 453-460 (2004).</li></ol>

The author has no conflicts of interest.

In living organisms, cell death is defined as apoptosis or necrosis25&#160;and can be accompanied by autophagy26. The difference between apoptotic and necrotic cells is that apoptosis is a form of programmed cell death and appears in normal cells, whereas necrosis occurs due to lethal conditions (e.g., accident, disease)27,28. Apoptosis can be detected using assay kits based on the TUNEL technique (detection ofDNA f...

Honeybees (Apis mellifera L.) are, among other wild pollinators, the most important pollinators of agricultural plants. Over thousands of years, the changing environment has influenced bees to adapt their morphology, physiology, behavior, and tolerance to several pathogens and parasites. Therefore, honeybees have developed a highly diverse range of species and subspecies around the globe1. These results are consistent with previous findings, that there is genetic variation in the honeybee&#39;s digestive tract structure, but also suggest that alterations of the midgut are due to environmental factors2,3.
The digestive tract of the honeybee has three main parts: foregut, midgut (ventriculus), and hindgut4. The ventriculus is an essential organ for the digestion of pollen and nectar/honey; in the hindgut, osmotic control takes place through absorption of water and ions2. The hypopharyngeal glands (HPGs) of honeybee workers are located in the head and synthesize and secrete royal jelly components to feed the brood, the queen, and members of the colony. Their size changes with age and tasks and depends on proper nutrition (quality pollen). Nurse workers aged 6 to 18 days perform brood rearing, and the size of HPGs increases5,6. In forager bees, the HPGs degenerate and only secrete enzymes that are important to convert the complex sugars into simple ones (&#945;-glucosidases, leucine arylamidase, invertase) in honey7.
Honeybees are exposed to several biotic and abiotic stressors8, and the digestive tract can be affected by several negative stimulants. The first barrier that protects the organism from pathogens is the peritrophic membrane in the midgut, which consists of intestinal mucosa to protect against pathogens4. The development and function of HPGs depend on diet, age, and colony condition9, and are affected by insecticides, acaricides10, and pathogens11,12,13. Acaricide residues in the hive due to varroa control treatment and pesticides from the environment affect forager bees and nurse bees14,15. The greatest threat to honeybee colonies is the mite Varroa destructor, both as a vector of viruses contributing to colony losses16&#160;and as a consumer of the host&#39;s fat body (an important vital organ in honeybees), which consequently affects the individual&#39;s body and colony functions17.
However, intensive farmland habitats can provide a short-term food supply for honeybees. Therefore, agri-environmental schemes should enhance the availability of honey flowers in agricultural landscapes18. To assess the morphology of different subspecies6,19,20,21&#160;or sublethal effects of these factors at the cell or tissue levels, especially midgut and HPGs, histological and immunohistochemical methods are practical and sufficiently accurate to be used in histology research in honeybees.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Propanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ApopTag Peroxidase kit (ApopTag Peroxidase In Situ Apoptosis Detection)</td>
			<td>Sigma-Aldrich</td>
			<td>S7100</td>
			<td>Assay B, https://www.sigmaaldrich.com/SI/en/product/mm/s7100?gclid=CjwKCA 
			jw7vuUBhBUEiwAEdu2pPanI9SE 
			j81ZTl-nLHEoxXAv7ViKwPA_QRx 
			H7fciMRNcYwR7lbPQbhoCqcQQA 
			vD_BwE; Positive controls included in S7101</td>
		</tr>
		<tr>
			<td>Covers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DeadEnd Colorimetric TUNEL system</td>
			<td>Promega</td>
			<td>G7360</td>
			<td>Assay A, https://worldwide.promega.com/products/cell-health-assays/apoptosis-assays/deadend-colorimetric-tunel-system/?catNum=G7360</td>
		</tr>
		<tr>
			<td>Dissecting microscope&#160; (for bee dissection)</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding cassette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EnVision System alkaline phosphatase kit</td>
			<td>Dako</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y Solution</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>alcoholic</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td>95% (or less pure), 90%, 80%</td>
		</tr>
		<tr>
			<td>Faramount mounting medium, aqueous</td>
			<td>Dako</td>
			<td></td>
			<td>mounting medium</td>
		</tr>
		<tr>
			<td>Flattening table</td>
			<td>Leica</td>
			<td>HI1220</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps&#160; (for bee dissection)</td>
			<td>Fine science tools</td>
			<td>11294-00</td>
			<td>Standard #4</td>
		</tr>
		<tr>
			<td>Formalin 10%</td>
			<td></td>
			<td></td>
			<td>Formaldehyde</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HistoChoice Clearing Agent</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>clearing agent</td>
		</tr>
		<tr>
			<td>Hydrogen peroxidase 3%</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>BioRad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insect pins&#160; (for bee dissection)</td>
			<td>Entosphinx</td>
			<td>44594</td>
			<td>Insect pins stainless steel &#8211; white, size 2</td>
		</tr>
		<tr>
			<td>ISCDDK, AP (In Situ Cell Death Detecteion Kit, Alkaline Phosphatase)</td>
			<td>Roche</td>
			<td>11684809910</td>
			<td>Assay C, https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/362/737/11684809910b 
			ul.pdf</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lab clock</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td></td>
			<td></td>
			<td>Box with the slides must be preserved in a plastic wrap to prevent dust</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Modular tissue embedding station</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 4%</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraplast</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipettes</td>
			<td></td>
			<td></td>
			<td>1.5 mL; 3 mL</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish&#160; (for bee dissection)</td>
			<td></td>
			<td></td>
			<td>Filled with condensation silicon&#160; (Xantoprene L blue and Universal liquid plus activator)</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Merck</td>
			<td>21627</td>
			<td></td>
		</tr>
		<tr>
			<td>Ringers&#39; solution&#160; (for bee dissection)</td>
			<td></td>
			<td></td>
			<td>7.5 g NaCL, 2.38 g Na2HPO4, 2.72 g KH2PO4, 1 L distilled water</td>
		</tr>
		<tr>
			<td>Scissors&#160; (for bee dissection)</td>
			<td>Fine science tools</td>
			<td>1406-09, 14061-09</td>
			<td>Straight and curved, 9 cm</td>
		</tr>
		<tr>
			<td>Universal liquid plus activator&#160; (for bee dissection)</td>
			<td>Kulzer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Watchmaker&#8217;s forceps (for bee dissection)</td>
			<td>Fine science tools</td>
			<td>91100-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Watercolor brush</td>
			<td></td>
			<td></td>
			<td>2x</td>
		</tr>
		<tr>
			<td>Xantoprene L blue&#160; (for bee dissection)</td>
			<td>Kulzer</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

histology basics and cell death detection in honeybee tissue

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather changes, various pesticides, pathogens, and malnutrition, mainly entering through the mouth and primarily affecting the digestive tracts of adult bees. To understand and prevent the effects of such external and internal stressors on honeybees, one useful research method is the immunohistochemical method. A basic protocol is described to prepare the midgut (ventriculus) and hypopharyngeal glands (HPGs) of adult bees for histological analysis. A detailed methodology is described to assess the level of cell damage and distinguish necrosis from programmed cell death (apoptosis) as a natural process of tissue regeneration. The results of adult honeybee treatment with oxalic acid and pesticides (insecticide and acaricide) and the determination of cell death in the ventriculus and HPGs are presented. The pros and cons of the methodology are also discussed.

Cell death detection in the midgut 
Newly emerged worker bees (Apis mellifera carnica) from the experimental apiary at the Agricultural Institute of Slovenia in Ljubljana were individually treated with 3% oxalic acid (OA)23. OA is frequently used in beekeeping for Varroa destructor control. After the treatment, the worker bees (three from each group) were immobilized on ice. The midgut was dissected and fixed it in 10% formalin. The tissue was then dehydrate...

Watch this Scientific Journal Video about Histology Basics and Cell Death Detection in Honeybee Tissue at JoVE.com

Histology Basics and Cell Death Detection in Honeybee Tissue

Immunohistochemical methods are useful in honeybee research to detect and assess the level of apoptosis and necrosis in the midgut and hypopharyngeal glands of adult bees.

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather ...

histology-basics-and-cell-death-detection-in-honeybee-tissue

Research

JoVE Journal

Biology

2.1K Views.  Agricultural Institute of Slovenia. Immunohistochemical methods are useful in honeybee research to detect and assess the level of apoptosis and necrosis in the midgut and hypopharyngeal glands of adult bees.

Video: Histology Basics and Cell Death Detection in Honeybee Tissue

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides vital information on bone turnover or bone formation and resorption. This has tremendous importance in a variety of clinical and research applications. It yields beautiful images1 and allows for techniques such as fluorochrome assessment and histomorphometry2. Fluorochrome analysis is a technique where fluorescent dyes that bind to calcium are injected at a particular time point, which allows for quantification of the amount of mineralisation at that given time. Histomorphometry is a process of bone quantification at the microscopic level. 
Performing undecalcified bone histology is technically challenging, particularly with large size specimens. It requires variations in technique from those used in standard paraffin embedded histology. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them. Specimen preparation, fixation and processing are achieved with a manner similar to other soft tissues, however due to the density and lower permeability of bone considerably longer fixation and processing times are required, often taking several weeks. Embedding is achieved using a supporting medium with similar or equal hardness and density to the bone such as methacrylate- based resins, but unlike paraffin infiltration and embedding, this is an irreversible step. Sectioning can be achieved by grinding which produces a thicker section, which is optimal for studies such as fluorochrome analysis. This is best achieved using a diamond blade on a macrotome. Alternatively, thinner sections can be produced for light microscopy and this is achieved using a sledge microtome with a very sharp blade. The sledge microtome provides the additional strength and stability required for large, hard blocks. Resin embedded sections can be stained with a variety of stains, which are demonstrated.

Undecalcified bone histology provides important information for a variety of clinical and research applications. It is technically challenging, particularly with large size specimens. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them.

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are analyzed. Established methods to detect cell division include direct visualization by continuous microscopy in cell culture, dilution of vital dyes such as carboxy&#64258;uorescein di-aetate succinimidyl ester (CFSE), immuno-detection of mitogenic antigens such as ki67 or PCNA, and thymidine analogues. Thymidine analogues can be detected by a variety of methods including radio-detection for tritiated thymidine, immuno-detection for bromo-deoxyuridine (BrdU), chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU), and chemical detection for ethinyl-deoxyuridine (EdU). We have derived a strategy to detect sequential incorporation of different thymidine analogues (CldU and IdU) into tissues of adult mice. Our method allows investigators to accurately quantify two successive rounds of cell division. By optimizing immunostaining protocols our approach can detect very low dose thymidine analogues administered via the drinking water, safe to administer to mice for prolonged periods of time. Consequently, our technique can be used to detect cell turnover in very long-lived tissues. Optimal immunofluoresent staining results can be achieved in multiple tissue types, including pancreas, skin, gut, liver, adrenal, testis, ovary, thyroid, lymph node, and brain. We have also applied this technique to identify oncogenic transformation within tissues. We have further applied this technique to determine if transit-amplifying cells contribute to growth or renewal of tissues. In this sense, sequential administration of thymidine analogues represents a novel approach for studying the origins and survival of cells involved in tissue homeostasis.

Immunofluorescent Detection of Two Thymidine Analogues CldU and IdU in Primary Tissue

We have derived a strategy to detect sequential incorporation of thymidine analogues (CldU and IdU) into tissues of adult mice to quantify two successive rounds of cell division. This strategy is useful to detect cell turnover of long-lived tissues, oncogenic transformation, or transit-amplifying cells.

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are ...

A &#946;-glucuronidase (GUS) Based Cell Death Assay

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative positive or negative regulators of cell death. In this system, N. benthamiana leaves are co-infiltrated with a 35S driven expression cassette containing the gene to be analyzed, and the GUS vector pCAMBIA 2301 using Agrobacterium strain LBA4404 as a vehicle. Because live cells are required for GUS expression to occur, loss of GUS activity is expected when this marker gene is co-expressed with positive regulators of cell death. Equally, increased GUS activity is observed when anti-apoptotic genes are used compared to the vector control. As shown below, we have successfully used this system in our lab to analyze both pro- and anti-death players. These include the plant anti-apoptotic Bcl-2 Associated athanoGene (BAG) family, as well as, known mammalian inducers of cell death, such as BAX. Additionally, we have used this system to analyze the death function of specific truncations within proteins, which could provide clues on the possible post-translational modification/activation of these proteins. Here, we present a rapid and sensitive plant based method, as an initial step in investigating the death function of specific genes.

A &#946;-glucuronidase GUS Based Cell Death Assay

Programmed cell death assays commonly used in mammalian systems such as DNA laddering or TUNEL assays, are often difficult to reproduce in plants. In combination with a GUS reporter system, we propose a rapid, plant based transient assay to analyze the potential death properties of specific genes.

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Manipulating the Murine Lacrimal Gland

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for vision. In the human a single LG resides in the orbit above the lateral end of each eye delivering tears to the ocular surface through 3 - 5 ducts. The mouse has three pairs of major ocular glands, the most studied of which is the exorbital lacrimal gland (LG) located anterior and ventral to the ear. Similar to other glandular organs, the LG develops through the process of epithelial branching morphogenesis in which a single epithelial bud within a condensed mesenchyme undergoes multiple rounds of bud and duct formation to form an intricate interconnected network of secretory acini and ducts. This elaborate process has been well documented in many other epithelial organs such as the pancreas and salivary gland. However, the LG has been much less explored and the mechanisms controlling morphogenesis are poorly understood. We suspect that this under-representation as a model system is a consequence of the difficulties associated with finding, dissecting and culturing the LG. Thus, here we describe dissection techniques for harvesting embryonic and post-natal LG and methods for ex vivo culture of the tissue.

The lacrimal gland (LG) is a branching organ that produces the aqueous components of tears necessary for maintaining vision and ocular health. Here we describe murine LG dissection and ex vivo culture techniques to decipher signaling pathways involved in LG development.

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...

Histology Basics and Cell Death Detection in Honeybee Tissue

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Chapters in this video

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue

A β-glucuronidase (GUS) Based Cell Death Assay

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Manipulating the Murine Lacrimal Gland

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation